Bacteria of the genus Lactobacillus are widely used as oral probiotics due to their putative health benefits. In this study, we compared the colonization ability of two Lactobacillus strains that were identified as autochthonous to the human gastrointestinal tract (Lactobacillus reuteri ATCC PTA 6475 (MM4-1a) and (Lactobacillus mucosae FSL-04) with that of an allochthonous strain (Lactobacillus acidophilus DDS-1). Colonization ability was tested in a single-blinded, cross-over study, with twelve human subjects. The test strains were quantified in fecal samples by two independent methods, selective plating and molecular typing and quantitative real time PCR. The study revealed that the two autochthonous strains (L. reuteri ATCC PTA 6475 and L. mucosae FSL-04) reached higher population levels in fecal samples and were recovered more frequently from subjects compared to the allochthonous strain (L. acidophilus DDS-1). All three strains became undetectable 8 days after the end of consumption with one exception, showing that persistence of all three strains remains short term in most individuals. In conclusion, this study showed that autochthonous Lactobacillus strains can be established more efficiently, albeit temporarily, in the human gastrointestinal tract, suggesting that evolutionary and ecological characteristics could be valuable criteria for the selection of probiotic strains.